SOLUBLE ErbB3 METHOD OF DETECTION

ABSTRACT

The present invention discloses a method using human soluble ErbB3, for example p85-sErbB3, as a negative regulator of heregulin-stimulated ErbB2, ErbB3, and ErbB4 activation. The present invention also discloses p85-sErbB3 binding to heregulin with an affinity comparable to that of full-length ErbB3, and competitively inhibiting high affinity heregulin binding to ErbB2/ErbB3 heterodimers on the cell surface of breast carcinoma cells. The present invention also uses p85-sErbB3 to inhibit heregulin-induced phosphorylation of ErbB2, ErbB3, and ErbB4 in cells, as a negative regulator of heregulin-stimulated signal transduction, and as a block for cell growth. The present invention is also directed to nucleic acids and expression vectors encoding p85-sErbB3, host cells harboring such expression vectors, and methods of producing the protein. The present invention discloses a method of therapeutically treating human malignancies associated with heregulin-mediated cell growth such as breast and prostate cancer.

This application is a divisional of U.S. application Ser. No. 10/159,353filed May 31, 2002, which claims priority to U.S. ProvisionalApplication No. 60/294,824 filed May 31, 2001, both of which areincorporated herein by reference.

The disclosed invention was made with the support of a grant from theNational Cancer Institute (CA85133). The United States Government hascertain rights in the invention.

FIELD OF INVENTION

Embodiments of the present invention generally pertain to methods andtherapeutics that relate to soluble ErbB3 proteins (sErbB3), includingp85-sErbB3, p45-sErbB3 and other isoforms of sErbB3, wherein said sErbB3protein binds to heregulins and antagonizes heregulin-stimulatedactivation of the ErbB receptors and blocks the cell proliferativeactivity thereof. The present invention also is directed to expressionvectors encoding an sErbB3 protein, including p85-sErbB3, p45-sErbB3 andother isoforms of ErbB3, host cells harboring such expression vectors,methods of preparing such proteins, and methods and systems utilizingsuch proteins for the treatment of conditions associated with undesiredheregulin stimulation.

BACKGROUND OF THE INVENTION

The following background information is provided to assist the reader tounderstand the invention disclosed below and the environment in which itwill typically be used. The terms used herein are not intended to belimited to any particular narrow interpretation unless clearly statedotherwise, either expressly or impliedly, in this document.

The heregulins (also called neuregulins, neu differentiation factor(NDF), acetylcholin receptor inducing activity (ARIA), glial growthfactors (GGFs)) are a family of growth factors that activate members ofthe ErbB/EGF receptor family (Holmes, Sliwkowski et al. 1992; Peles,Bacus et al. 1992; Wen, Peles et al. 1992; Falls, Rosen et al. 1993;Marchionni, Goodearl et al. 1993). Isoforms of heregulins, all of whicharise from splice variants of a single gene, NRG-1 (neuregulin-1), havebeen cloned and classified into the α and β subgroups based onstructural differences in their EGF binding domains (Holmes, Sliwkowskiet al. 1992).

ErbB-mediated signal transduction exerted by heregulins has beenimplicated in the regulation of diverse biological events includingSchwann cell differentiation, neural regulation of skeletal muscledifferentiation, heart development, and proliferation anddifferentiation of normal and malignant breast epithelial cells (Alroyand Yarden 1997; Sundaresan, Penuel et al. 1999). Research has shownthat breast carcinoma cells respond to heregulin through proliferation,differentiation, as well as morphogenesis. Carcinoma cells expressingheregulin are hormone-independent and correlated with the ability formetastasis in experimental studies.

ErbB3 is a transmembrane glycoprotein encoded by the c-erbB3 gene(Kraus, Issing et al. 1989; Plowman, Whitney et al. 1990). The ErbB3receptor belongs to the ErbB family which is composed of four growthfactor receptor tyrosine kinases, known as ErbB1/EGFR, ErbB2/Neu, ErbB4,as well as ErbB3. ErbB3 and ErbB4 are receptors for heregulins and ErbB2is a coreceptor (Carraway and Burden 1995). These receptors arestructurally related and include three functional domains: anextracellular ligand-binding domain, a transmembrane domain, and acytoplasmic tyrosine kinase domain (Plowman, Culouscou et al. 1993). Theextracellular domain can be further divided into four subdomains (I-IV),including two cysteine-rich regions (II and IV) and two flanking regions(I and III). The ErbB3 is unusual among receptor tyrosine kinases inthat its catalytic domain is defective. Despite its lack of intrinsiccatalytic activity, ErbB3 is an important mediator of heregulinresponsiveness. Heregulin binding induces ErbB3 to associate with othermembers of the ErbB family to form heterodimeric receptor complexes.ErbB3 then transactivates the kinase of its partner receptor whichinitiates a variety of cytoplasmic signaling cascades.

The ErbB3 receptor is important in regulating cellular growth anddifferentiation. Particular attention has focused on the role of ErbB3as a coreceptor of ErbB2 in the area of cancer research. Transgenic micethat have been engineered to overexpress heregulin in mammary glandshave been reported to exhibit persistent terminal end buds and, overtime, to develop mammary adenocarcinomas (Krane and Leder 1996). ErbB3expression studies on tumor tissues and on cell lines show frequentco-expression of ErbB2 and ErbB3 receptors (Alimandi, Heidaran et al.1995; Meyer and Birchmeier 1995; Robinson, He et al. 1996; Siegel, Ryanet al. 1999). In addition, both ErbB2 and ErbB3 are activated in mammarytumors formed in transgenic mice harboring only the activated form ofErbB2 (Siegel, Ryan et al. 1999). Many cell lines used for experimentaltumor formation studies are either estrogen-dependent (MCF-7 and T47D,the low ErbB2 expressers) or estrogen-independent (SKBR3, high ErbB2expressers). However, these cell lines do not exhibit metastaticphenotypes. When MCF-7 cells are transfected to overexpress ErbB2, MCF-7cells gain estrogen-independent phenotype, however, they nevermetastasize. On the other hand, the MCF-7 cells overexpressing heregulingain metastatic phenotype, suggesting heregulin's active role inmetastasis (Hijazi, Thompson et al. 2000; Tsai, Hornby et al. 2000).

Five alternate ErbB3 transcripts arise from read-through of an intronand the use of alternative polyadenylation signals (Lee and Maihle 1998;Katoh, Yazaki et al. 1993). Using 3′-RACE the inventors have isolatedfour novel c-erbB-3 cDNA clones of 1.6, 1.7, 2.1, and 2.3 kb from ahuman ovarian carcinoma-derived cell line (Lee and Maihle 1998).p85-sErbB3 of 543 amino acids (aa), encoded by a 2.1 kb alternatec-erbB3 transcript (cDNA clone R31F), is composed of subdomains Ithrough III and the first third of subdomain IV, and has a unique 24amino acid carboxy-terminal sequence. p45-sErbB3 of 312 aa, encoded by a1.7 kb alternate c-erbB3 transcript (cDNA clone R2F) contains subdomainsI, II, and a portion of subdomain III of the extracellular domain ofErbB-3 followed by two unique glycine residues. p50-sErbB3 of 381 aa,encoded by a 1.6 kb alternate c-erbB3 transcript (cDNA clone RiF)contains subdomains I, II, and a portion of subdomain III of theextracellular domain of ErbB-3 followed by 30 unique amino acids.p75-sErbB3 of 515 aa, encoded by a 2.3 kb alternate c-erbB3 transcript(cDNA clone R35F), is composed of subdomains I through III, and has aunique 41 amino acid carboxy-terminal sequence (FIG. 1) (Lee and Maihle1998).

Using various recombinant forms of EGFR, it has been shown thatefficient inhibition of full-length EGFR activation by dominant-negativeheterodimerization occurs only when these deletion mutants retain thetransmembrane domain in addition to the extracellular domain (Redemann,Holzmann et al. 1992). Similarly, a recombinant dominant-negative ErbB3mutant with a deleted cytoplasmic domain but which retains itstransmembrane domain can inhibit full-length ErbB2 and ErbB3 activation(Ram, Schelling et al. 2000). In contrast, in avian tissues, expressionof a naturally occurring sEGFR/ErbB1 inhibits TGFα dependenttransformation (Flickinger, Maihle et al. 1992). An aberrant solubleEGFR secreted by the A431 human carcinoma cell line also has beenreported to inhibit the kinase activity of purified full-length EGFR ina ligand-independent manner (Basu, Raghunath et al. 1989). In no case dothese soluble EGFR/ErbB1 receptors function as antagonists through highaffinity ligand-binding. Similarly, herstatin, a naturally occurringsoluble ErbB2 protein which inhibits ErbB2 activation appears tofunction by blocking ErbB2 dimerization (Doherty, Bond et al. 1999).

The soluble ErbB3 protein, specifically the p85-sErbB3 and p45 sErbB3isoforms, is unique among other naturally occurring ErbB receptors inthat it binds specifically to heregulin with high affinity and inhibitsits binding to cell surface receptors and consequently inhibitsheregulin-induced activation of the receptors and their downstreameffectors. Thus sErbB3, specifically p85-sErbB3 and p45-sErbB3, can beused as therapeutic reagents for heregulin-induced malignancies such asmammary and prostate tumors.

Heretofore, production and purification methods for, therapeutic usesof, and useful compositions containing, this protein, referred to hereinas p85-sErbB3 have not been available.

SUMMARY OF THE INVENTION

Embodiments of the present invention pertain to several novel isolatedand purified nucleic acids which encode soluble isoforms of ErbB3.Preferred embodiments of this aspect of the invention are nucleic acidsequences which specifically encode a soluble form of ErbB3 whose aminoacid sequence comprises the sequence of SEQ ID NO:2 or SEQ ID NO: 4. Therelated nucleic acid embodiments comprise SEQ ID NO: 1 and SEQ ID NO: 3.

Particular embodiments of the present invention relate to isoforms ofsErbB3 that bind to HRG with high affinity and effectively blockheregulin (HRG) binding to cell surface receptors. Even moreparticularly, the embodiments of the present invention relate to the useof p85-sErbB3 to bind to HRG with high affinity and substantially blockHRG binding to cell surface receptors. Embodiments of the presentinvention also pertain to the diagnosis and treatment of cancer cellsusing p85-sErbB3 and other sErbB3 isoforms.

A preferred embodiment of the present invention pertains to anexpression vector, such as a plasmid or virus, containing the isolatedcDNA encoding p85-sErbB3 or other sErbB3 isoforms, as well as a cell,either eukaryotic or prokaryotic, containing the expression vector.

Embodiments of the present invention also pertain to a process forproducing the p85-sErbB3 isoform and other sErbB3 isoforms, whichcomprises the steps of ligating the isolated DNA into an expressionvector capable of expressing the isolated DNA in a suitable host;transforming the host with the expression vector; culturing the hostunder conditions suitable for expression of the isolated DNA andproduction of the p85-sErbB3 protein or other sErbB3 isoforms, andisolating the protein from the host. The host cell may be a prokaryote,or a eukaryote.

Further embodiments of the present invention relate to polyclonal ormonoclonal antibodies directed against unique p85-sErbB3 or other sErbB3isoform epitopes. Particular embodiments relate to polyclonal andmonoclonal antibodies specific to p85-sErbB3 generated using aC-terminal unique sequence of the p85-sErbB3 as an antigen. Theaffinity-purified antibody can be used to detect p85-sErbB3 usingimmunoblot analysis and other detection methods.

Another embodiment of the invention relates to a system and method ofdetecting p85-sErbB3 and other sErbB3 isoforms in a mammalian biologicalspecimen which is selected from the group consisting of fluids(including blood, serum, plasma, urine and ascites), tissues, and theirderivatives. A particular embodiment of the present invention pertainsto immunoprecipitation followed by immunoblot analysis to detectp85-sErbB3 using anti-ErbB3 antibodies.

Yet another embodiment of this invention relates to a vector for genetherapy, comprising a nucleic acid molecule having i) a transcriptionregulatory sequence; and ii) a second sequence coding for p85-sErbB3 orother sErbB3 isoforms under transcriptional control of the transcriptionregulatory sequence; and a delivery vehicle for delivering the nucleicacid molecule.

Other aspects, embodiments, features and advantages of the presentinvention will be apparent from reading the description of the followingpreferred embodiments.

BRIEF DESCRIPTION OF THE FIGURES AND DEFINITIONS

FIG. 1. Diagram of soluble ErbB3 (sErbB3) proteins. ErbB3 is composed ofa 19 amino acid (aa) signal peptide sequence that is cleaved (gray box),an extracellular ligand-binding domain (aa1-620), a transmembrane domain(aa 621-646; indicated as TM), and an intracellular domain (aa647-1323). The extracellular domain of the receptor can be furtherdivided into four subdomains (I-IV), as noted in the text. The alternatec-erbB3 transcripts arise from read-through of an intron and the use ofalternative polyadenylation signals. p45-sErbB3 contains theamino-terminal 310 amino acids of ErbB3 and two unique carboxy-terminalamino acid residues. p50-sErbB3 contains the amino-terminal 351 aminoacids of ErbB3 and 30 unique carboxy-terminal amino acid residues.p75-sErbB3 contains the amino-terminal 474 amino acids of ErbB3 and 41unique carboxy-terminal amino acid residues. p85-sErbB3 contains theamino-terminal 519 amino acids of ErbB3 and 24 unique carboxy-terminalamino acid residues. The carboxy-terminal unique sequences are denotedas black boxes.

FIG. 2. p45-sErbB3 and p85-sErbB3 in conditioned media can blockHRG-induced activation of ErbB3. (A) p45-sErbB3 and p85-sErbB3 in theconcentrated conditioned media were detected by Western blotting usingan anti-ErbB3 antibody recognizing the extracellular region of ErbB3.Increasing volumes (5, 10, 20 μl; left to right) of the concentratedconditioned media (×15) were loaded on an SDS-PAGE gel. (B) and (C) TheBa/F3 (ErbB2+ErbB3) cells were stimulated with HRGα (panel B) and HRGβ(panel C) with or without the concentrated conditioned media for 10 minat room temperature prior to lysis. ErbB3 was immunoprecipitated with ananti-ErbB3 antibody from equal amounts of total protein, subjected toSDS-PAGE, and analyzed by Western blotting using an anti-phosphotyrosineantibody (αPY). Filters were stripped and reprobed with anti-ErbB3antibody recognizing the intracellular region of ErbB3.

FIG. 3. p85-sErbB3 binds to HRG. (A) HRGβ was crosslinked to p85-sErbB3(25 mM) with BS³ after incubating in the presence of 50 nM ¹²⁵I-HRGβwithout or with increasing concentrations (0.16, 0.32, 0.64, 1.25 μM) ofunlabeled HRGβ. Insulin (1.25 μM) was used as a negative control. Thearrowhead indicates a 90 kDa complex of ¹²⁵I-HRGβ and p85-sErbB3. (B)and (C) Binding analysis of ¹²⁵I-HRG to p85-sErbB3 and ErbB3-IgG fusionprotein. Binding assays were performed in a 96-well plate format asdescribed below in more detail in the Examples. Binding results wereanalyzed by using Scatchard method and by plotting the displacement of¹²⁵I-HRGβ₁₇₇₋₂₄₄ binding by unlabeled HRGβ₁₇₇₋₂₄₄ (Inset).

FIG. 4. Inhibition of HRGβ binding by p85-sErbB3 and by 2C4, amonoclonal antibody specific for ErbB2. T47D cells were incubated withthe indicated concentrations of p85-sErbB3 and 2C4 at room temperaturefor 30 min. ¹²⁵I-HRGβ₁₇₇₋₂₄₄ (0.1 nM) was then added and bindingreactions were performed as described below in more detail in theExamples. ¹²⁵I-HRGβ₁₇₇₋₂₄₄ bound to the cell surface was measured usinga gamma counter.

FIG. 5. p85-sErbB3 blocks HRG-induced activation of ErbB2 and ErbB3 inthe Ba/F3 (ErbB2+ErbB3) cells. Cells were untreated or stimulated withHRGβ₁₋₂₄₁ alone or HRGβ₁₋₂₄₁ plus purified p85-sErbB3 for 10 min at roomtemperature. Receptor phosphorylation levels and ErbB2 and ErbB3receptor levels were determined by anti-ErbB2 (A) and anti-ErbB3 (B)immunoprecipitation followed by Western blotting as described in FIG. 2.

FIG. 6. p85-sErbB3 blocks HRG-induced activation of ErbB proteins andtheir downstream activators MAPK, PI3K (p85), and Akt. (A) p85-sErbB3blocks HRG-induced activation of ErbB2, ErbB3, and ErbB4 in T47D andMCF7 breast carcinoma cells. Serum-starved cells were stimulated with noHRGβ, HRGβ alone, or 6 nM HRGβ plus 36 nM p85-sErbB3 for 10 min at roomtemperature. Receptor phosphorylation levels and ErbB2, ErbB3, and ErbB4receptor levels were determined by immunoprecipitation followed byWestern blotting. (B) p85-sErbB3 inhibits HRG-induced association ofPI3K (p85) with ErbB3 and activation of MAPK and Akt in T47D cells.Cells were treated with 1 nM HRGβ and 10 mM p85-sErbB3 for 10 min or 30min and analyzed for activation of ErbB3. Association of PI3K (p85) withErbB3 was analyzed by immunoprecipitation of cell lysates using ananti-ErbB3 antibody followed by Western blotting of anti-PI3K (p85)antibody. Activation of MAPK and Akt was examined by Western blotting ofcell lysates using antibodies specific to phospho-MAPK and phospho-Akt.

FIG. 7. p85-sErbB3 inhibits cell growth stimulation by HRG. MCF7 cellswere trypsinized, washed, and plated at a density of 5,000 (squares) or8,000 cells/well (triangles) in 96-well plates with increasingconcentrations of HRGβ in serum-free medium and growth was measuredafter 3 days (inset). MCF7 cells were trypsinized, washed, incubatedwith p85-sErbB3 for 30 min, and plated with or without 0.4 nM HRGβ inserum-free medium. At 40 nM (a 100-fold molar excess to HRGβ) in thepresence of HRGβ, p85-sErbB3 inhibited cell growth by 75% and 90%, atdensities of 5,000 and 8,000 cells/well, respectively, whereas the sameconcentration of p85-sErbB3 did not affect cell growth in the absence ofHRGβ. The data presented are the mean±standard deviation of sixreplicates. This experiment was repeated three times and the resultsshown represent all three trials.

DEFINITIONS

As used herein, the term “soluble” ErbB3 (sErbB3) means that the ErbB3polypeptide is found in a form that is not anchored to the membrane of acell, i.e., a portion of the sErbB3 is not found physically embedded inthe lipid bilayer which comprises the cell membrane in the organism ofits origin. As used herein, the term “biological activity” of a peptideof the invention is defined to mean a polypeptide comprising a subunitof a peptide having SEQ ID NO:2, or a variant thereof, which has atleast about 10%, preferably at least about 50%, and more preferably atleast about 90% of the activity of a peptide having SEQ ID NO:2. Theactivity of a peptide of the invention can be measured by methods wellknown in the art including, but not limited to, the ability to bindheregulins, or the ability of the peptide to elicit a sequence-specificimmune response when the peptide is administered to an organism, e.g.,goat, sheep or mice.

The terms “recombinant nucleic acid” or “preselected nucleic acid,”e.g., “recombinant DNA sequence or segment” or “preselected DNA sequenceor segment” refer to a nucleic acid that has been derived or isolatedfrom any appropriate tissue source and that may be subsequentlychemically altered, typically in vitro, so that its sequence is notnaturally occurring, or corresponds to naturally occurring sequencesthat are not positioned as they would be positioned in a genome whichhas not been transformed with exogenous DNA. An example of preselectedDNA “derived” from a source, would be a DNA sequence that is identifiedas a useful fragment within a given organism and which is thenchemically synthesized in essentially pure form. An example of such DNA“isolated” from a source would be a useful DNA sequence that is excisedor removed from said source by chemical means, e.g., by the use ofrestriction endonucleases, so that it can be further manipulated, e.g.,amplified, for use in the invention, by the methodology of geneticengineering.

“Regulatory sequences” is defined to mean RNA or DNA sequences necessaryfor the expression, post-transcriptional modification, translation, andpost-translational modification of an operably linked coding sequence ina particular host organism. The control sequences that are suitable forprokaryotic cells, for example, include a promoter, and optionally anoperator sequence, and a ribosome binding site. Eukaryotic cells areknown to utilize promoters, stop sequences, enhancers, splicing, andpolyadenylation signal sequences, as well as glycosylation and secretorysignal sequences.

As used herein, the term “cell line” or “host cell” is intended to referto well-characterized homogenous, biologically pure populations ofcells. These cells may be eukaryotic cells that are neoplastic or whichhave been “immortalized” in vitro by methods known in the art, as wellas primary cells, or prokaryotic cells. The cell line or host cell ispreferably of mammalian origin, but cell lines or host cells ofnon-mammalian origin may be employed, including avian, plant, insect,yeast, fungal or bacterial sources. Generally, the preselected DNAsequence is related to a DNA sequence that is resident in the genome ofthe host cell but is not expressed, or not highly expressed, or,alternatively, over-expressed.

The terms “transfected” or “transformed” are used herein to include anyhost cell or cell line, the genome of which has been altered oraugmented by the presence of at least one preselected DNA sequence,which DNA is also referred to in the art of genetic engineering as“heterologous DNA,” “recombinant DNA,” “exogenous DNA,” “geneticallyengineered DNA,” “non-native DNA,” or “foreign DNA,” wherein said DNAwas isolated and introduced into the genome of the host cell or cellline by the process of genetic engineering. The host cells of thepresent invention are typically produced by transfection with a DNAsequence in a plasmid expression vector, a viral expression vector, oras an isolated linear DNA sequence. Preferably, the transfected DNA is achromosomally integrated recombinant DNA sequence, which comprises agene encoding an sErbB3 isoform, which host cell may or may not expresssignificant levels of autologous or “native” sErbB3.

DESCRIPTION OF THE INVENTION

Using various recombinant forms of EGFR, it has been shown thatefficient inhibition of full-length EGFR activation by dominant-negativeheterodimerization occurs only when these deletion mutants retain thetransmembrane domain and the extracellular domain. Similarly, arecombinant dominant-negative ErbB3 mutant with a deleted cytoplasmicdomain but which retains its transmembrane domain can inhibitfull-length ErbB2 and ErbB3 activation. In contrast, in avian tissues,expression of a naturally occurring sEGFR/ErbB1 inhibits TGFα dependenttransformation. An aberrant soluble EGFR secreted by the A431 humancarcinoma cell line also has been reported to inhibit the kinaseactivity of purified full-length EGFR in a ligand-independent manner.These soluble EGFR/ErbB1 receptors do not function as antagoniststhrough high affinity ligand-binding. Similarly, herstatin, a naturallyoccurring soluble ErbB2 protein which inhibits ErbB2 activation appearsto function by blocking ErbB2 dimerization; this inhibition is thoughtto be mediated via ligand-independent binding of herstatin to ErbB2. Incontrast, sErbB3, including p85-sErbB3 and p45-sErbB3, inhibitsHRG-induced stimulation of ErbB2, ErbB3, and ErbB4, at least in part, byneutralizing ligand activity through competitive binding. The presentinvention discloses that p85-sErbB3 is capable of binding to the cellsurface.

The physiological role of p85-sErbB3 in normal tissues also has not beenunderstood to date. As discussed in greater detail below, although amuch higher concentration (100-fold) was required to inhibit cellgrowth, a 10-fold molar excess of p85-sErbB3 was sufficient forinhibition of phosphorylation of ErbB receptors. At this ratio, a smallfraction of receptors are still activated and are sufficient for growthstimulation. It is known that the 2.1 kb transcript encoding p85-sErbB3is expressed at low levels compared to the full-length c-erbB3transcript in all cell lines and tissues examined to date, however,local expression of this transcript has been studied in detail. It is,therefore, plausible that p85-sErbB3 acts as a HRG antagonist locally ina tissue-specific and/or stage-specific manner, and related studies toexamine the distribution of p85-sErbB3 in selected tissues are currentlyunderway. Research in the field shows that local concentrations ofautocrine growth factors such as EGF are exquisitely regulated and donot travel far from the cell surface from which they are released. Inthis context, tightly regulated levels of local p85-sErbB3 expressionhave important consequences on cellular activities, such as HRG-mediatedcell growth. These consequences are even more dramatic in cancer cellswhere cell polarity is typically lost, resulting in deregulation ofnormal spatial and temporal control of growth factor:receptorinteractions.

The present invention provides several novel isolated and purifiednucleic acids which encode isoforms of ErbB3 and nucleic acids encodingengineered variants of these proteins. Preferred embodiments are nucleicacids which specifically encode isoforms of ErbB3 whose amino acidsequence comprises the sequence of SEQ ID NO: 2 and SEQ ID NO: 4. Thepresent invention also defines the biochemical and biologicalcharacteristics of a novel sErbB3 isoform designated p85-sErbB3.Embodiments of the present invention relate to the use of p85-sErbB3 asa unique HRG inhibitor because it can block HRG binding to cell surfacereceptors via binding to HRG with high affinity, thereby, inhibitingHRG-induced stimulation of ErbB2, ErbB3, and ErbB4. This inhibition issufficient to effectively block HRG-stimulated cell growth. These novelsErbB3 receptor isoforms, therefore, are potent modulators of HRGregulated cell proliferation and differentiation in normal humantissues, and as such provide an ideal candidate for the development ofnovel cancer therapeutics.

EXAMPLES AND PREFERRED EMBODIMENTS

Conditioned Media from Cells Expressing p45-sErbB3 and p85-sErbB3Inhibit HRG Activation of ErbB3. p45-sErbB3 and p85-sErbB3 are naturallyoccurring secreted products of the ErbB3 gene (Lee and Maihle 1998).p45-sErbB3 contains the amino-terminal 310 amino acids of ErbB3 and twounique carboxy-terminal amino acid residues. p85-sErbB3 contains theamino-terminal 519 amino acids of ErbB3 and 24 unique carboxy-terminalamino acid residues (See FIG. 1). To examine whether p45-sErbB3 andp85-sErbB3 can modulate HRG receptor activation cells stably transfectedwith these corresponding cDNA clones were isolated. These cells secretep45-sErbB3 and p85-sErbB3 into the culture medium (See FIG. 2A). Theconditioned medium from these cells was used as the source of p45-sErbB3or p85-sErbB3 in a series of preliminary experiments described below.

To test the ability of p45-sErbB3 and p85-sErbB3 to modulate aspects ofHRG-mediated ErbB receptor activation a clonal derivative of the Ba/F3cell line expressing exogenous ErbB2 and ErbB3 was stimulated with HRGαEGF domain₁₇₇₋₂₄₁ (HRGα) and HRGβ1₁₇₆₋₂₄₆ (HRGβ) in the absence orpresence of concentrated conditioned media containing p45-sErbB3 andp85-sErbB3. As shown in FIG. 2, HRGβ was at least 20-fold more effectivethan HRGα in stimulating ErbB3 tyrosine phosphorylation. Conditionedmedia containing sErbB3 inhibited HRGα-stimulated ErbB3 activation by40% (p45-sErbB3) and 80% (p85-sErbB3) at 1 μg/ml HRGα, as determined bydensitometric analysis. However, at a higher concentration (2 μg/ml),conditioned media containing p85-sErbB3 decreased activation by 30%,although inhibition by conditioned media containing p45-sErbB3 wasnegligible (See FIG. 2A). In the presence of conditioned mediumcontaining either p45-sErbB3 or p85-sErbB3, ligand stimulation of ErbB3tyrosine phosphorylation was decreased by 60% and 90%, respectively, atboth 50 and 100 ng/ml HRGβ (See FIG. 2C). These data indicate thatp85-sErbB3 inhibited ErbB3 phosphorylation in response to both HRGα andHRGβ more effectively than p45-sErbB3, although the concentration ofp85-sErbB3 used in these studies was lower than that of p45-sErbB3 (FIG.2A).

Purification of p85-sErbB3. p85-sErbB3 was isolated from a concentratedconditioned medium of cells stably transfected with a cDNA clone R31Fencoding p85-sErbB3 and was purified in two steps. The first step waslectin affinity chromatography with a Con A column (Sigma). The boundp85-sErbB3 was washed with column buffer (10 mM Tris-HCl, pH 7.5, 150 mMNaCl, 1 mM MnCl₂, and 1 mM CaCl₂) and eluted using column buffercontaining 1 M a-methyl D-mannoside, then dialyzed against 20 mMTris-HCl, pH 7.5 overnight. The second step of purification wasaccomplished using a Mono Q®, an ion exchanger for resolution ofproteins and peptides, ion exchange FPLC®, i.e., a microprocessorcontrolled, solvent delivery apparatus used in purification ofbiologically active compounds column (Pharmacia). The bound p85-sErbB3was eluted from the column with 0-500 mM NaCl gradient containing 20 mMTris-HCl, pH 7.5. Samples taken from each step were subjected toSDS-PAGE in duplicate and analyzed by Coomassie staining and by Westernblot using anti-ErbB3 236 antibody recognizing the extracellular domainof the ErbB3 (Lee and Maihle 1998). The final p85-sErbB3 pool washomogeneous on SDS-PAGE, and the identity of the purified protein wasconfirmed by Western blot analysis. Purified preparations of p85-sErbB3were used in all subsequent experiments.

p85-sErbB3 Binds to HRG with High Affinity. Previous reports based theassignation of the subdomain boundaries of the ErbB3 extracellulardomain on the subdomain boundaries of EGFR (Lee and Maihle 1998) asdefined by the genomic structure of avian ErbB1 (Callaghan, Antczak etal. 1993). Accordingly, p85-sErbB3 is composed of subdomains I throughIII and includes the first 45 amino acids of subdomain IV (aa 1-519),and a unique twenty-four amino acid sequence at the carboxy-terminus.Binding studies using heregulins indicate that subdomains I and II arerequired for heregulin binding (Singer, Landgraf et al. 2001). On theother hand, for EGF binding to EGFR subdomains I and III are low andhigh affinity binding sites, respectively (Lax, Bellot et al. 1989).Because p85-sErbB3 contains both subdomains I through III the presentinvention determined that p85-sErbB3 would bind to heregulins.

Direct binding between p85-sErbB3 and radiolabeled HRGβ was examinedusing the chemical crosslinker BS³. As shown in FIG. 3A, a proteincomplex of 90 kDa was formed between p85-sErbB3 and ¹²⁵I-HRGβ. Formationof this complex could be inhibited by addition of excess cold HRGβ butnot by addition of excess insulin, indicating that p85-sErbB3 binding toHRGβ is specific and that purified preparations of p85-sErbB3 arebiologically active. An analysis of ¹²⁵I-HRGβ₁₇₇₋₂₄₄ binding toimmobilized p85-sErbB3 was then performed using an ErbB3-IgG homodimeras a positive control. As shown in FIG. 3, p85-sErbB3 binds toHRGβ₁₇₇₋₂₄₄ with a K_(D) of 3.0±0.2 nM. In comparison, ErbB3-IgG bindsto HRGβ₁₇₇₋₂₄₄ with a K_(D) of 4.7±0.2 mM. These results demonstratethat p85-sErbB3 binds to HRGβ₁₇₇₋₂₄₄ with an affinity similar to that ofthe extracellular domain of ErbB3. Based on the results of these twocomplementary experimental approaches, the present invention establishesthe use of p85-sErbB3 to bind to HRG with an affinity equivalent to theaffinity of HRG for the full-length extracellular domain of ErbB3.

p85-sErbB3 Inhibits Binding of HRG to Receptors on the Cell Surface. Thepresent invention also discloses the use of p85-sErbB3 to effectivelylimit binding of heregulin to cell surface receptors in the breastcarcinoma cell line T47D. This cell line expresses all four ErbBreceptors at moderate levels. Cells were incubated with varyingconcentrations of p85-sErbB3 in the presence of ¹²⁵I-labeledHRGβ₁₇₇₋₂₄₄. Simultaneously, a separate group of cells was incubatedwith ¹²⁵I-HRGβ₁₇₇₋₂₄₄ in the presence of varying concentrations of 2C4,a monoclonal antibody specific for the ErbB2 extracellular domain(Lewis, Lofgren et al. 1996). As shown by the inhibition curves (SeeFIG. 4), p85-sErbB3 and 2C4 inhibit HRGβ₁₇₇₋₂₄₄ binding to cell surfacereceptors with similar IC₅₀ values (0.45±0.03 nM and 0.55±0.03 nM,respectively) although the mechanism of inhibition by these twomolecules is distinct. Although 2C4 inhibits heregulin binding to cellsurface receptors by blocking ErbB2-ErbB3 heterodimerization via bindingto the ErbB2 extracellular domain (Fitzpatrick, Pisacane et al. 1998),p85-sErbB3 inhibited receptor activation, at least in part, by competingfor heregulin binding to the cell surface.

p85-sErbB3 Blocks HRG-Induced Activation of ErbB2, ErbB3, and ErbB4. Thepresent invention also examined the ability of p85-sErbB3 to modulateHRG-stimulated receptor activation in the Ba/F3 (ErbB2+ErbB3) cell lineusing purified p85-sErbB3. This allowed an analysis of the mechanism ofp85-sErbB3 mediated inhibition in a quantitative manner. As shown inFIG. 5, when Ba/F3 (ErbB2+ErbB3) cells were treated with p85-sErbB3 at a10-fold molar excess over HRGβ₁₋₂₄₁, ErbB3 phosphorylation levels werereduced to basal levels. A similar level of receptor inhibition also wasapparent when either a 2.5- or 5-fold molar excess of p85-sErbB3 wasused in these experiments. Exogenous addition of p85-sErbB3 alsoinhibited HRG-induced ErbB2 activation. p85-sErbB3 blocked HRGstimulation whether the cells were treated with the EGF-like domain ofHRG (HRGα or HRGβ), as shown in FIG. 2, or with HRGβ₁₋₂₄₁ (See FIG. 5),suggesting that inhibition by p85-sErbB3 occurs, at least in part,through a direct interaction between p85-sErbB3 and the EGF-like domainof HRG. Cells treated with the same concentration of p85-sErbB3 but notstimulated with HRG did not exhibit altered ErbB2 or ErbB3 tyrosinephosphorylation, or show any change in the level of either ErbB2 orErbB3 expression, suggesting that p85-sErbB3 does not function as a“ligand” for these receptors.

To examine whether exogenous addition of p85-sErbB3 exerts the sameinhibitory effect on endogenously expressed ErbB receptors, and todetermine whether p85-sErbB3 could modulate other members of the EGFreceptor family, the activity of p85-sErbB3 in two breast carcinoma celllines, i.e., T47D and MCF7, was tested. As shown in FIG. 6A, addition ofp85-sErbB3 (at a 6-fold molar excess relative to HRGβ) inhibitedHRG-induced activation of ErbB2, ErbB3, and ErbB4 in both the T47D andMCF7 cell lines. In contrast, at least in these two cell lines whichexpress low EGFR levels, EGFR phosphorylation remained at basal level incells treated with HRGβ regardless of whether p85-sErbB3 was present ornot. Similarly, EGF-induced phosphorylation of EGFR or ErbB2, or, to alesser degree, phosphorylation of ErbB3, was not decreased byp85-sErbB3. These results demonstrate that inhibition by p85-sErbB3 isspecific for HRG-induced activation of ErbB2, ErbB3, and ErbB4.

It is notable that in the T47D cells, a decrease in ErbB2, ErbB3, andErbB4 protein levels following HRG stimulation was observed. In MCF7cells a decrease in ErbB3 levels also was apparent when HRG was added tothe culture medium (See FIG. 6A). It has been reported that thepolyclonal ErbB3 antibody specific to the carboxy-terminal 17 aa used inthis study preferentially recognizes non-phosphorylated ErbB3 on Westernblots (Vartanian, Goodearl et al. 1997). Thus, when T47D or MCF7 cellsare stimulated with HRG, a significant fraction of ErbB3 isphosphorylated, and, therefore, undetectable with this particular ErbB3antibody. The anti-ErbB antibodies used in these experiments recognizethe carboxy-terminal 17 aa (ErbB3) and 18 aa (ErbB2 and ErbB4) sequencesof these receptors. Each of these sequences contains one tyrosineresidue. Immunoblot detection by the anti-ErbB2 and ErbB4 antibodiesused in this study, therefore, may reflect either the level of receptorexpression or the unphosphorylated fraction of these receptors.

p85-sErbB3 Inhibits Activation of Downstream Effectors of HRG.HRG-stimulated activation of ErbB2, ErbB3, and ErbB4 leads to activationof two major signal transduction pathways: the PI3K pathway and the MAPKpathway (Wallasch, Weiss et al. 1995). The present invention testedwhether p85-sErbB3 could inhibit activation of these two downstreameffector pathways in T47D cells. Specifically, the present inventionexamined activation of MAPK and Akt by analyzing the phosphorylationlevels of these proteins, and examined the ability of p85phosphatidylinositide 3-kinase (“PI3K”) to interact with ErbB3 followingHRGβ treatment. In the presence of p85-sErbB3 (10-fold molar excessrelative to HRGβ), tyrosine phosphorylation of ErbB3 was reduced tobasal levels. In the same cell population, addition of exogenousp85-sErbB3 abrogated the phosphorylation of both MAPK and Akt asdetermined by Western blot analysis, and inhibited ErbB3's associationwith p85 PI3K (See FIG. 6B). These results further demonstrate thatp85-sErbB3 can inhibit the activation of ErbB2, ErbB3, and ErbB4, andthis inhibition affects the activation of downstream signaling moleculessuch as MAPK, Akt, and PI3K.

p85-sErbB3 Inhibits HRG-stimulated Cell Growth. The present inventionalso discloses the inhibition of HRG-induced phosphorylation of ErbBreceptors by p85-sErbB3 as correlated with the modulation of HRG'sbiological effects. Specifically, a cell growth assay using MCF7 cellsstimulated with HRGβ was performed and showed that, within theconcentration range tested, growth of this cell line was dose-dependent(See FIG. 7). It was observed that at a concentration of 0.4 nM HRGβ thecell growth rate was half of the rate observed at saturating levels ofHRGβ. In cell cultures grown in the presence of 0.4 nM HRGβ andp85-sErbB3 (a 100-fold molar excess relative to HRGβ), p85-sErbB3inhibited cell growth by 75% and 90%, at densities of 5,000 and 8,000cells/well, respectively, whereas the same concentration of p85-sErbB3did not affect cell growth in the absence of HRGβ (See FIG. 7). Thus,the present invention discloses the use of p85-sErbB3 as a potentinhibitor of HRG-dependent breast carcinoma cell growth in vitro.

REFERENCES

-   Alimandi, M., M. Heidaran, et al. (1995). “Cooperative signaling of    ErbB3 and ErbB2 in neoplastic transformation and human mammary    carcinomas.” Oncogene 10: 1813-1821.-   Alroy, I. and Y. Yarden (1997). “The ErbB signaling network in    embryogenesis and oncogenesis: signal diversification through    combinatorial ligand-receptor interactions.” FEBS Lett. 410(1):    83-86.-   Basu, A., M. Raghunath, et al. (1989). “Inhibition of tyrosine    kinase activity of the epidermal growth factor (EGF) receptor by a    truncated receptor form that binds to EGF: role for interreceptor    interaction in kinase regulation.” Mol. Cell. Biol. 9(2): 671-677.-   Callaghan, T., M. Antczak, et al. (1993). “A complete description of    the EGF-receptor exon structure: implication in oncogenic activation    and domain evolution.” Oncogene 8: 2939-2948.-   Carraway, K. L. I. and S. J. Burden (1995). “Neuregulins and their    receptors.” Current Opinion in Neurobiology 5: 606-612.-   Falls, D. L., K. M. Rosen, et al. (1993). “ARIA, a protein that    stimulates acetylcholine receptor synthesis, is a member of the neu    ligand family.” Cell 72(5): 801-15.-   Fitzpatrick, V. D., P. I. Pisacane, et al. (1998). “Formation of a    high affinity heregulin binding site using the soluble extracellular    domains of ErbB2 with ErbB3 or ErbB4.” FEBS Lett. 431(1): 102-106.-   Flickinger, T. W., N. J. Maihle, et al. (1992). “An alternatively    processed mRNA from the avian c-erbB gene encodes a soluble,    truncated form of the receptor that can block ligand-dependent    transformation.” Mol. Cell. Biol. 12(2): 883-893.-   Hijazi, M. M., E. W. Thompson, et al. (2000). “Heregulin regulates    the actin cytoskeleton and promotes invasive properties in breast    cancer cell lines.” International Journal of Oncology 17(4): 629-41.-   Holmes, W. E., M. X. Sliwkowski, et al. (1992). “Identification of    heregulin, a specific activator of p185erbB2.” Science 256(5060):    1205-1210.-   Krane, I. M. and P. Leder (1996). “NDF/heregulin induces persistence    of terminal end buds and adenocarcinomas in the mammary glands of    transgenic mice.” Oncogene 12(8): 1781-1788.-   Kraus, M. H., W. Issing, et al. (1989). “Isolation and    characterization of ERBB3, a third member of the ERBB/epidermal    growth factor receptor family: evidence for overexpression in a    subset of human mammary tumors.” PNAS 86: 9193-9197.-   Lax, I., F. Bellot, et al. (1989). “Functional analysis of the    ligand binding site of EGF-receptor utilizing chimeric chicken/human    receptor molecules.” EMBO J. 8(2): 421-427.-   Lee, H. and N. J. Maihle (1998). “Isolation and characterization of    four alternate c-erbB3 transcripts expressed in ovarian    carcinoma-derived cell lines and normal human tissues.” Oncogene    16(25): 3243-3252.-   Lewis, G. D., J. A. Lofgren, et al. (1996). “Growth regulation of    human breast and ovarian tumor cells by heregulin: Evidence for the    requirement of ErbB2 as a critical component in mediating heregulin    responsiveness.” Cancer Res. 56(6): 1457-1465.-   Marchionni, M. A., A. D. Goodearl, et al. (1993). “Glial growth    factors are alternatively spliced erbB2 ligands expressed in the    nervous system.” Nature 362(6418): 312-8.-   Meyer, D. and C. Birchmeier (1995). “Multiple essential functions of    neuregulin in development [see comments] [published erratum appears    in Nature 1995 Dec. 14; 378(6558):753].” Nature 378(6555): 386-390.-   Peles, E., S. S. Bacus, et al. (1992). “Isolation of the neu/HER-2    stimulatory ligand: a 44 kd glycoprotein that induces    differentiation of mammary tumor cells.” Cell 69(1): 205-16.-   Plowman, G. D., J. M. Culouscou, et al. (1993). “Ligand-specific    activation of HER4/p180erbB4, a fourth member of the epidermal    growth factor receptor family.” Proc. Natl. Acad. Sci. USA 90(5):    1746-1750.-   Plowman, G. D., G. S. Whitney, et al. (1990). “Molecular cloning and    expression of an additional epidermal growth factor receptor-related    gene.” Proceedings of the National Academy of Sciences of the United    States of America 87(13): 4905-4909.-   Ram, T. G., M. E. Schelling, et al. (2000). “Blocking HER-2/HER-3    function with a dominant negative form of HER-3 in cells stimulated    by heregulin and in breast cancer cells with HER-2 gene    amplification.” Cell Growth Differ. 11(3): 173-183.-   Redemann, N., B. Holzmann, et al. (1992). “Anti-oncogenic activity    of signalling-defective epidermal growth factor receptor mutants.”    Mol. Cell. Biol. 12(2): 491-498.-   Robinson, D., F. He, et al. (1996). “A tyrosine kinase profile of    prostate carcinoma.” Proc. Natl. Acad. Sci. USA 93(12): 5958-5962.-   Siegel, P. M., E. D. Ryan, et al. (1999). “Elevated expression of    activated forms of Neu/ErbB-2 and ErbB-3 are involved in the    induction of mammary tumors in transgenic mice: implications for    human breast cancer.” EMBO Journal 18(8): 2149-2164.-   Singer, E., R. Landgraf, et al. (2001). “Identification of a    heregulin binding site in HER3 extracellular domain.” Journal of    Biological Chemistry 276(47): 44266-74.-   Sundaresan, S., E. Penuel, et al. (1999). “The biology of human    epidermal growth factor receptor 2.” Curr. Oncol. Report 1: 16-22.-   Tsai, M. S., A. E. Hornby, et al. (2000). “Expression and function    of CYR61, an angiogenic factor, in breast cancer cell lines and    tumor biopsies.” Cancer Research 60(20): 5603-7.-   Vartanian, T., A. Goodearl, et al. (1997). “Axonal Neuregulin    Signals Cells of the Oligodendrocyte Lineage through Activation of    HER4 and Schwann Cells through HER2 and HER3.” J. Cell Biol. 137:    211.-   Wallasch, C., F. U. Weiss, et al. (1995). “Heregulin-dependent    regulation of HER2/neu oncogenic signaling by heterodimerization    with HER3.” EMBO J. 14(17): 4267-4275.-   Wen, D., E. Peles, et al. (1992). “Neu differentiation factor: a    transmembrane glycoprotein containing an EGF domain and an    immunoglobulin homology unit.” Cell 69(3): 559-72.

REFERENCES

-   Alimandi, M., M. Heidaran, et al. (1995). “Cooperative signaling of    ErbB3 and ErbB2 in neoplastic transformation and human mammary    carcinomas.” Oncogene 10: 1813-1821.-   Alroy, I. and Y. Yarden (1997). “The ErbB signaling network in    embryogenesis and oncogenesis: signal diversification through    combinatorial ligand-receptor interactions.” FEBS Lett. 410(1):    83-86.-   Basu, A., M. Raghunath, et al. (1989). “Inhibition of tyrosine    kinase activity of the epidermal growth factor (EGF) receptor by a    truncated receptor form that binds to EGF: role for interreceptor    interaction in kinase regulation.” Mol. Cell. Biol. 9(2): 671-677.-   Callaghan, T., M. Antczak, et al. (1993). “A complete description of    the EGF-receptor exon structure: implication in oncogenic activation    and domain evolution.” Oncogene 8: 2939-2948.-   Carraway, K. L. I. and S. J. Burden (1995). “Neuregulins and their    receptors.” Current Opinion in Neurobiology 5: 606-612.-   Doherty, J. K., C. Bond, et al. (1999). “The HER-2/neu receptor    tyrosine kinase gene encodes a secreted autoinhibitor.” Proc. Natl.    Acad. Sci. USA 96(19): 10869-10874.-   Falls, D. L., K. M. Rosen, et al. (1993). “ARIA, a protein that    stimulates acetylcholine receptor synthesis, is a member of the neu    ligand family.” Cell 72(5): 801-15.-   Fitzpatrick, V. D., P. I. Pisacane, et al. (1998). “Formation of a    high affinity heregulin binding site using the soluble extracellular    domains of ErbB2 with ErbB3 or ErbB4.” FEBS Lett. 431(1): 102-106.-   Flickinger, T. W., N. J. Maihle, et al. (1992). “An alternatively    processed mRNA from the avian c-erbB gene encodes a soluble,    truncated form of the receptor that can block ligand-dependent    transformation.” Mol. Cell. Biol. 12(2): 883-893.-   Hijazi, M. M., E. W. Thompson, et al. (2000). “Heregulin regulates    the actin cytoskeleton and promotes invasive properties in breast    cancer cell lines.” International Journal of Oncology 17(4): 629-41.-   Holmes, W. E., M. X. Sliwkowski, et al. (1992). “Identification of    heregulin, a specific activator of p185erbB2.” Science 256(5060):    1205-1210.-   Katoh, M., Y. Yazaki, et al. (1993). “c-erbB3 gene encodes secreted    as well as transmembrane receptor tyrosine kinase.” Biochem.    Biophys. Res. Commun. 192(3): 1189-1197.-   Krane, I. M. and P. Leder (1996). “NDF/heregulin induces persistence    of terminal end buds and adenocarcinomas in the mammary glands of    transgenic mice.” Oncogene 12(8): 1781-1788.-   Kraus, M. H., W. Issing, et al. (1989). “Isolation and    characterization of ERBB3, a third member of the ERBB/epidermal    growth factor receptor family: evidence for overexpression in a    subset of human mammary tumors.” Proc. Natl. Acad. Sci. USA 86:    9193-9197.-   Lax, I., F. Bellot, et al. (1989). “Functional analysis of the    ligand binding site of EGF-receptor utilizing chimeric chicken/human    receptor molecules.” EMBO J. 8(2): 421-427.-   Lee, H. and N. J. Maihle (1998). “Isolation and characterization of    four alternate c-erbB3 transcripts expressed in ovarian    carcinoma-derived cell lines and normal human tissues.”Oncogene    16(25): 3243-3252.-   Lewis, G. D., J. A Lofgren, et al. (1996). “Growth regulation of    human breast and ovarian tumor cells by heregulin: Evidence for the    requirement of ErbB2 as a critical component in mediating heregulin    responsiveness.” Cancer Res. 56(6): 1457-1465.-   Marchionni, M. A, A D. Goodearl, et al. (1993). “Glial growth    factors are alternatively spliced erbB2 ligands expressed in the    nervous system.” Nature 362(6418): 312-8.-   Meyer, D. and C. Birchmeier (1995). “Multiple essential functions of    neuregulin in development [see comments] [published erratum appears    in Nature 1995 Dec. 14; 378(6558):753].” Nature 378(6555): 386-390.-   Peles, E., S. S. Bacus, et al. (1992). “Isolation of the neu/HER-2    stimulatory ligand: a 44 kd glycoprotein that induces    differentiation of mammary tumor cells.” Cell 69(1): 205-16.-   Plowman, G. D., J. M. Culouscou, et al. (1993). “Ligand-specific    activation of HER4/p180erbB4, a fourth member of the epidermal    growth factor receptor family.” Proc. Natl. Acad. Sci. USA 90(5):    1746-1750.-   Plowman, G. D., G. S. Whitney, et al. (1990). “Molecular cloning and    expression of an additional epidermal growth factor receptor-related    gene.” Proc. Natl. Acad. Sci. USA 87(13): 4905-4909.-   Ram, T. G., M. E. Schelling, et al. (2000). “Blocking HER-2/HER-3    function with a dominant negative form of HER-3 in cells stimulated    by heregulin and in breast cancer cells with HER-2 gene    amplification.” Cell Growth Differ. 11(3): 173-183.-   Redemann, N., B. Holzmann, et al. (1992). “Anti-oncogenic activity    of signalling-defective epidermal growth factor receptor mutants.”    Mol. Cell. Biol. 12(2): 491-498.-   Robinson, D., F. He, et al. (1996). “A tyrosine kinase profile of    prostate carcinoma.” Proc. Natl. Acad. Sci. USA 93(12): 5958-5962.-   Siegel, P. M., E. D. Ryan, et al. (1999). “Elevated expression of    activated forms of Neu/ErbB-2 and ErbB-3 are involved in the    induction of mammary tumors in transgenic mice: implications for    human breast cancer.” EMBO Journal 18(8): 2149-2164.-   Singer, E., R. Landgraf, et al. (2001). “Identification of a    heregulin binding site in HER3 extracellular domain.” Journal of    Biological Chemistry 276(47): 44266-74.-   Sundaresan, S., E. Penuel, et al. (1999). “The biology of human    epidermal growth factor receptor 2.” Curr. Oncol. Report 1: 16-22.-   Tsai, M. S., A. E. Hornby, et al. (2000). “Expression and function    of CYR61, an angiogenic factor, in breast cancer cell lines and    tumor biopsies.” Cancer Research 60(20): 5603-7.-   Vartanian, T., A. Goodearl, et al. (1997). “Axonal Neuregulin    Signals Cells of the Oligodendrocyte Lineage through Activation of    HER4 and Schwann Cells through HER2 and HER3.” J. Cell Biol. 137:    211.-   Wallasch, C., F. U. Weiss, et al. (1995). “Heregulin-dependent    regulation of HER2/neu oncogenic signaling by heterodimerization    with HER3.” EMBO J. 14(17): 4267-4275.-   Wen, D., E. Peles, et al. (1992). “Neu differentiation factor: a    transmembrane glycoprotein containing an EGF domain and an    immunoglobulin homology unit.” Cell 69(3): 559-72.    p85-sErbB3 Protein

1-13. (canceled)
 14. A method for determining the concentration of ansErbB3 in a biological sample from a mammal, the method comprising: a)obtaining a biological sample from the mammal; b) contacting an amountof a first purified antibody that specifically reacts with a firstepitope of the sErbB3 with the biological sample to be tested, whereinthe first purified antibody is modified with a first labeling moiety; c)contacting the biological sample with an amount of a second purifiedantibody that specifically reacts with a second epitope of the sErbB3,wherein the second purified antibody is modified with a second labelingmoiety, and wherein the second purified antibody does not competitivelyinhibit the binding of the first purified antibody; and d) detecting theco-presence of the first and second antibodies to determine theconcentration of the sErbB3.
 15. The method of claim 14 wherein thebiological sample is chosen from the group consisting of urine, ascites,blood, serum, plasma, tissue and derivatives thereof.
 16. The method ofclaim 14 wherein the first or second epitope of the sErbB3 is at least aportion of the sErbB3 unique carboxy terminal region.
 17. The method ofclaim 14 wherein the first or second epitope of the sErbB3 is at least aportion of the sErbB3 extracellular ligand binding domain.
 18. Themethod of claim 14 wherein the sErbB3 is selected from the groupconsisting of p85-sErbB3, p45-sErbB3, p50-sErbB3, and p75-sErbB3.